1. Field of the Invention
The present invention relates generally to the TT virus and to methods of use thereof. More particularly, the present invention relates to nucleic acid primers useful for detection of the TT virus, use of the TT virus as a vector, use of the TT virus for human and veterinary diagnostics, and use of the TT virus for testing prior to transplantation or xenotransplantation. Additionally, the present invention includes use of TT virus sequence diversity as a means of monitoring viral transmission between individuals.
2. Background of the Invention
Recently, a novel human DNA virus was isolated from the serum of a Japanese patient (initials T.T.) with cryptogenic hepatitis (Nishizawa et al., Biochem Biophys Res Commun 241:92-97 (1997)). Utilizing PCR, TT virus (TTV) was detected in sera from three of five patients with non-A to GBV-C hepatitis. Subsequently, the nearly complete nucleotide sequence of the TTV genome, encompassing 3739 bases, and a more sensitive PCR assay for the detection of virus in serum were reported (Okamoto et al., Hepatol. Res. 10:1-16 (1998)). In addition, based upon sensitivity to single-strand but not double-strand-specific endonucleases, the virus appeared to possess a single-stranded DNA genome. Data presented regarding the size of the genome, its single-strandedness, and resistance to detergents, suggested that TTV was similar to the parvoviruses (Okamoto et al., Hepatol. Res. 10:1-16 (1998)). However, the buoyant density in CsCl (1.31-1.32 g/ml) was lower than that reported for the parvoviruses.
Several PCR studies have been performed to assess the prevalence of this virus in various populations. One assay described by Okamoto et al. (Okamoto et al., Hepatol. Res. 10:1-16 (1998)) detected TTV DNA in hemophiliacs (68%), intravenous drug abusers (40%), patients on maintenance hemodialysis (46%) and those with cryptogenic hepatitis and/or chronic liver disease (46-48%). Further, TTV infection in Japanese normal blood donors was found to be 12%. The rates of TTV infection in the United Kingdom have recently been reported at 1.9% (19 of 1000 blood donors) (Simmonds et al., The Lancet 352:191-194 (1998)) using two distinct primers sets and 10% (3 of 30 healthy controls) (Naoumov et al., The Lancet 352:195-197 (1998)) using the PCR strategy of Okamoto (Okamoto et al., Hepatol. Res. 10:1-16 (1998)). Both of these reports identified TTV DNA in patients at risk for acquiring parenterally transmitted viruses (27-39%) and/or in patients with hepatitis (19-22%). These studies suggest that TTV can be transmitted via blood or blood products and may also be associated with some cases of cryptogenic hepatitis.
The preliminary epidemiological studies of TTV described above utilized several different first generation PCR primer pairs. Desai et al. (Desai et al., J. Infect. Dis. in press:(1999)) compared the sensitivities of two first generation TTV PCR primers sets and demonstrated that the majority of TTV-positive samples were detected by only one of the two primer sets. Thus, previous reports that utilized a single PCR primer pair may have significantly underestimated the true prevalence of the virus. Second generation PCR assays for TTV appear to confirm the underestimation of TTV prevalence. Specifically, a PCR assay described by Takahashi et al (Takahashi et al., Hepatol. Res. 12:233-239 (1998)) that was 10 to 100 times more sensitive than the assay described by Okamoto et al (Okamoto et al., Hepatol. Res. 10:1-16 (1998)) found TTV present in 92 of 100 healthy individuals who visited a Japanese hospital for routine health screening. Therefore, TTV prevalence in the normal Japanese population appears to be much higher than the 12% originally reported.
The high rate of TTV carriers in the normal population may not be compatible with an exclusive parenteral transmission route. A possible fecal-oral transmission route was suggested by a study that demonstrated the presence of TTV in the feces of infected humans (Okamoto et al., J. of Med. Virol. 56:128-132 (1998)). Additional non-parenteral routes of infection may explain the high prevalence of TTV infection in healthy individuals. Finally, based upon limited prevalence studies and the high rates of TTV in the normal populations (Charlton et al., Hepatology 28:839-842 (1998; Naoumov et al., The Lancet 352:195-197 (1998; Simmonds et al., The Lancet 352:191-194 (1998)), the association between TTV infection and human hepatitis is questionable.
The detection of TTV in test samples can be enhanced by the use of DNA amplification assays that utilize DNA oligomers as primers, since the amount of DNA target nucleotides present in a test sample may be in minute amounts. Methods for amplifying and detecting a target nucleic acid sequence that may be present in a test sample are well-known in the art. Such methods include the polymerase chain reaction (PCR) described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the ligase chain reaction (LCR) described in European Patent Application EP-A-320 308, gap LCR (GLCR) described in European Patent Application EP-A-439 182 and U.S. Pat. No. 5,427,930 which is incorporated herein by reference, multiplex LCR described in International Patent Application No. WO 93/20227, and the like. These methods have found widespread application in the medical diagnostic field as well as in the fields of genetics, molecular biology and biochemistry.
It would be advantageous to provide DNA oligomer primers derived from TTV and diagnostics, and test kits which utilize these primers. Such primers could greatly enhance the ability to more accurately detect TTV infections, and track the virus"" route of transmission.
In addition to the advantages of viral detection, viruses have the potential to serve as vectors for purposes such as expression of cloned genes in culture and development of treatments for disease through gene therapy. Viruses that have been developed into vectors include those with DNA genomes such as adeno-associated virus (see, e.g., Muzyczka, N., Current Topics in Microbiol. and Immunol. 158:97-129 (1992) and Kotin, R. M., Human Gene Therapy 5:793-801 (1994)), adenovirus (see, e.g., Haj-Ahmad et al., J. Virol. 57:264-274 (1986) and Berkner, K. L., BioTechniques 6:619-629 (1988)), herpes virus (see, e.g., Breakefield et al., New Biol. 3:203-218 (1991) and Wolfe et al., Nature Genetics 1:379-384 (1992)) and papovavirus (see, e.g., Grossi et al., Arch. Virol. 102:275-283 (1988) and Milanesi et al., Mol Cell. Biol. 4:1551-1560 (1984)), and those with RNA genomes such as modified retroviruses (see, e.g., Gazit et al., Journal of Virology 60:19-28 (1986) and Palmer et al., Proc. Natl. Acad. Sci. USA 84:1055-1059 (1987)).
DNA viruses with small genomes, such as TTV, typically encode relatively few proteins and rely on the host cell to provide most replication and expression functions, thereby reducing the complexity of their interaction with the host cell. Furthermore, TTV infection does not appear to be associated with any disease, as is evidenced by its presence in nearly 100% of some human, normal populations. Its high prevalence also suggests that infection occurs readily, and that re-infection is common, as is implied by co-infections with multiple strains. All these traits are desirable in a gene therapy vector, which should be uncomplicated, non-pathogenic, easily delivered and have the potential for multiple treatments, or for being maintained over extended periods of time.
Comparison of numerous TTV genomes has demonstrated a high sequence diversity and an apparent lack of geographic localization. This implies either a high mutation rate, due to a low fidelity replicase, or an ancient virus family that has undergone extensive evolutionary drift. Researchers of HIV have used its sequence diversity as a basis for epidemiological studies and to demonstrate specific transmission of a viral infection from one individual to another. Likewise, the diversity of TTV may help establish the primary route of infection, and benefit investigations, such as forensics, that attempt to demonstrate contact between individuals.
All U.S. patents and publications are herein incorporated in their entirety by reference.
The present invention includes primers of probes specific for TT virus (TTV). These primers or probes are represented by SEQ ID NO:29, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71. Of particular interest within the above grouping are SEQ ID NOS:60-71.
Additionally, the present invention encompasses a method for detecting the presence of TTV target nucleotides which may be present in a test sample. This method comprises the steps of:(a) contacting a test sample suspected of containing a target TTV nucleotide sequence with a TTV primer pair consisting of: 1) SEQ ID NO:60 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, to form a reaction mixture which generates a product; (b) contacting the reaction mixture with a TTV primer pair consisting of: 1) SEQ ID NO:62 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, wherein the nucleotide sequence of the selected primer hybridizes with the product of the reaction mixture of (a); and (c) detecting the presence of the TTV target nucleotide in the test sample. The primer pair of step (a) consists of, for example SEQ ID NO:60 and SEQ ID NO:61, and the primer pair of step (b) consists of for example, SEQ ID NO:62 and SEQ ID NO:63.
Furthermore, the present invention also includes a method for detecting the presence of TTV target nucleotides which may be present in a test sample comprising the steps of: (a) contacting a test sample suspected of containing a target TTV nucleotide sequence with a TTV primer pair consisting of: 1) SEQ ID NO:64 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, to form a reaction mixture which generates a product; (b) contacting the reaction mixture with a TTV primer pair consisting of: 1) SEQ ID NO:66 and 2) a primer selected from the group consisting of SEQ ID SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, wherein the nucleotide sequence of the selected primer hybridizes with the product of the reaction mixture of (a); and (c) detecting the presence of the TTV target nucleotide in the test sample. The primer pair of step (a) consists of, for example SEQ ID NO:64 and SEQ ID NO:65, and the primer pair of step (b) consists of, for example, SEQ ID NO:66 and SEQ ID NO: 67.
Moreover, the present invention also encompasses a method for detecting the presence of TTV target nucleotides which may be present in a test sample. This method comprises the steps of: (a) contacting a test sample suspected of containing a target TTV nucleotide sequence with a TTV primer pair consisting of: 1) SEQ ID NO:68 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO;67, SEQ ID NO:69 and SEQ ID NO:71, to form a reaction mixture which generates a product; (b) contacting the reaction mixture with a TTV primer pair consisting of: 1) SEQ ID NO:70 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, wherein the nucleotide sequence of the selected primer hybridizes with the product of the reaction mixture; and (c) detecting the presence of the target TTV nucleotide in the test sample. The primer pair of step (a) consists of, for example, SEQ ID NO:68 and SEQ ID NO:69, and the primer pair of step (b) consists of, for example, SEQ ID NO:70 and SEQ ID NO:71. In any of the above methods, the test sample may be isolated from a human or an animal; thus, the methods may be used for both human and veterinary diagnostic purposes.
Additionally, the present invention includes a test kit for detecting target TTV nucleotides in a test sample, comprising: (a) a container containing a primer pair specific for a TTV target nucleotide, wherein said primer pair consists of 1) SEQ ID NO:60 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71; and (b) a container containing a primer pair specific for TTV, wherein said primer pair consists of 1) SEQ ID NO:62 and 2) a primer selected from the group consisting of is SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO;69 and SEQ ID NO:71. The primer pair of (a) consists of, for example, SEQ ID NO:60 and SEQ ID NO:61, and said primer pair of (b) consists of SEQ ID NO:62 and SEQ ID NO:63.
The present invention also encompasses a test kit for detecting target TTV nucleotides in a test sample, comprising: (a) a container containing a primer pair specific for a TTV target nucleotide, wherein the primer pair consists of 1)SEQ ID NO:64 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65. SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71; (b) a container containing a primer pair specific for TTV, wherein the primer pair consists of 1) SEQ ID NO:66 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71. The primer pair of (a) consists of, for example, SEQ ID NO:64 and SEQ ID NO:65, and the primer pair of (b) consists of, for example, SEQ ID NO:66 and SEQ ID NO:67.
Additionally, the present invention includes a test kit for detecting target TTV nucleotides in a test sample, comprising: (a) a container containing a primer pair specific for a TTV target nucleotide, wherein the primer pair consists of: 1) SEQ ID NO:68 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71; (b) a container containing a primer pair specific for TTV, wherein the primer pair consists of: 1) SEQ ID NO:70 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71. The primer pair of (a) consists of, for example, SEQ ID NO:68 and SEQ ID NO:69, and the pair of (b) consists of, for example, SEQ ID NO:70 and SEQ ID NO:71. Any of the test samples may be isolated from a human or an animal.
Furthermore, the present invention also encompasses a TTV-based vector comprising: 1) a promoter; 2) a heterologous DNA sequence; and 3) a nucleotide sequence encoding TTV, a fragment of the nucleotide sequence or a complement of the nucleotide sequence or the fragment, wherein the heterologous DNA sequence is operably linked to the promoter. The promoter may be derived from TTV or from a heterologous source. The heterologous DNA sequence may encode a polynucleotide sequence that is complementary to a targeted RNA sequence. For example, the heterologous DNA sequence may encodes protein. The vector may be capable of being packaged into TTV particles for stable maintenance or expression of said heterologous DNA sequence.
The invention also includes a host cell comprising the above vector. The host cell may be eukaryotic.
Additionally, the invention includes a method of expressing the heterologous DNA sequence or a product encoded by the heterologous DNA sequence, in a host, comprising introducing the vector into a host for a time and under conditions sufficient for expression of the heterologous DNA sequence or product encoded thereby.
The invention also includes a method of detecting transmission of TTV from one individual to another comprising the steps of: (a) obtaining a biological sample from an individual having TTV; (b) isolating a TTV DNA sequence from the biological sample; (c) obtaining a biological sample from a second individual having TTV; (d)isolating a TTV DNA sequence from the biological sample of the second individual; (e) comparing the TTV DNA sequence of the first individual with the TTV DNA sequence of the second individual, identity between the DNA sequence of the first individual and the DNA sequence of the second individual indicating transmission of TTV from one individual to the other.
The invention also includes a method of determining TTV-infection in a tissue or organ prior to transplantation or xenotransplantation of the tissue or organ comprising the steps of:
(a) contacting a biological sample suspecting of containing a TTV target nucleotide sequence, from a potential donor animal, with a TTV primer pair represented by SEQ ID NO:60 and SEQ ID NO:61 to form a first reaction mixture; (b) contacting said reaction mixture with a TTV primer pair represented by SEQ ID NO:62 and SEQ ID NO:63 in order to form a second reaction mixture; and (c) detecting the presence of the TTV target nucleotide in the test sample, presence of the nucleotide indicating TTV-infection in the biological sample and in the tissue or organ. The invention also includes a method of determining TTV-infection in a tissue or organ prior to transplantation or xenotransplantation of the tissue or organ comprising the steps of: (a) contacting a biological sample suspecting of containing a TTV target nucleotide sequence, from a potential donor animal, with a TTV primer pair represented by SEQ ID NO:64 and SEQ ID NO:65, to form a first reaction mixture; (b) contacting the reaction mixture with a TTV primer pair represented by SEQ ID NO:66 and SEQ ID NO:67; and c) detecting the presence of the TTV target nucleotide in the test sample, presence of the nucleotide indicating TTV-infection in the biological sample and in the tissue or organ.
Additionally, a method of determining TTV-infection in a tissue or organ prior to transplantation or xenotransplantation of said tissue or organ comprising the steps of:
(a) contacting a biological sample suspecting of containing a TTV target nucleotide sequence, from a potential donor animal, with a TTV primer pair represented by SEQ ID NO:68 and SEQ ID NO:69, to form a first reaction mixture;
(b) contacting said reaction mixture with a TTV primer pair represented by SEQ ID NO:70 and SEQ ID NO:71; and
(c) detecting the presence of the TTV target nucleotide in said test sample, presence of said nucleotide indicating TTV-infection in said biological sample and in said tissue or organ. In the above-mentioned methods, the biological sample may be selected from the group consisting of blood, tissue and an organ.
Additionally, the invention includes a method of detecting the presence of target TTV nucleotides in a test sample, comprising the steps of:
(a) contacting a test sample suspecting of containing a target TTV nucleotide with a primer pair represented by SEQ ID NO:60 and SEQ ID NO:61, to form a reaction mixture; (b) contacting said reaction mixture with at least one TTV probe selected from the group consisting of SEQ ID NO:62 and SEQ ID NO:63; and (c) detecting the presence of said target TTV nucleotide in said test sample. In yet another embodiment of the method, the test sample may be contacted with a primer pair represented by SEQ ID NO:64 and SEQ ID NO:65 and the resulting reaction mixture contacted with at least one TTV probe selected from the group consisting of SEQ ID NO:66 and SEQ ID NO:67. In yet another embodiment, the test sample may be contacted with primer pair SEQ ID NO:68 and SEQ ID NO:69 and the resulting reaction mixture contacted with at least one TTV probe selected from the group consisting of SEQ ID NO:70 and SEQ ID NO:71. In the above-mentioned embodiments, at least one TTV probe may be conjugated to a detectable signal-generating compound. Such a compound is selected from the group consisting of a chemiluminescent compound, fluorescein and an enzyme. In the alternative, the TTV probe may be conjugated to an antibody.
The invention also includes a method of detecting TTV target nucleotides which may be present in a test sample comprising contacting the test sample suspected of containing a target TTV nucleotide sequence with a TTV primer pair consisting of 1) a primer selected from the group consisting of: SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and SEQ ID NO:70, and 2) a primer selected from the group consisting: SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71, to form a reaction mixture which generates a product. In another embodiment, the method further comprises the steps of a) contacting the reaction mixture with a TTV primer pair consisting of: 1) a primer selected from the group consisting of: SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and SEQ ID NO:70, wherein the nucleotide sequence of the selected primer hybridizes with the product of the reaction mixture and 2) a primer selected from the group consisting: SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71, wherein the nucleotide sequence of the selected primer hybridizes with the product of the reaction mixture, and b) detecting the presence of the TTV target nucleotide in the test sample.
The present invention provides novel TT virus (TTV) DNA oligomer primers and probes. These DNA primers and probes are identified as SEQUENCE ID NOS. 60-71.
The present invention also provides an assay for detecting the presence of TTV in a test sample, which comprises (a) contacting a test sample suspected of containing a target TTV DNA sequence with a pair of TTV primers selected from the group consisting of the pair of SEQUENCE ID NOS. 60 and 61, followed by the pair of SEQUENCE ID NOS. 62 and 63; or the pair of SEQUENCE ID NOS. 64 and 65, followed by the pair of SEQUENCE ID NOS. 66 and 67; or the pair of SEQUENCE ID NOS. 68 and 69, followed by the pair of SEQUENCE ID NOS. 70 and 71, and (b) detecting the presence of the target DNA in the test sample. The TTV primers can be conjugated to a signal generating compound. This signal generating compound is selected from the group consisting of a chemiluminescent compound, a fluorescein compound and an enzyme. The reaction can be performed on a solid phase. Each primer can be attached to a different hapten such as adamantane and carbazole.
Also provided is a test kit for detecting target TTV DNA in a test sample, comprising (a) a container containing a TTV primer, wherein the primer is selected from the group consisting of the pair of SEQUENCE ID NOS. 60 and 61, followed by the pair of SEQUENCE ID NOS. 62 and 63; or the pair of SEQUENCE ID NOS. 64 and 65, followed by the pair of SEQUENCE ID NOS. 66 and 67; or the pair of SEQUENCE ID NOS. 68 and 69, followed by the pair of SEQUENCE ID NOS. 70 and 71, and (b) a container containing a detection reagents. The TTV primers can be conjugated to a detectable signal generating compound. This signal generating compound is selected from the group consisting of a chemiluminescent compound, a fluorescein compound and an enzyme. The reaction can be performed on a solid phase. Each primer primer can be attached to a different hapten such as adamantane and carbazole.
Also provided is the proposed use of the TTV genome, or parts thereof, to construct a vector for expression of cloned genes in culture or in gene therapy treatment. The vector can consist of the entire viral genome, either modified or wild type. It can also consist of parts of the genome such as the replication origin, specific genes, promoters or other control elements either by themselves or in conjunction with non-TTV sequences. A vector family is also proposed. The family would consist of identical sequences except for variable region(s) that prohibit re-infection of a previously infected host. The variable region(s) might encode epitopes from TTV isolates that do not show shared immunity, thus allowing multiple or prolonged treatment protocols.
Further provided is the use of TTV genomic diversity as a traceable marker to follow transmission of the virus between individuals, such traceability to be used in epidemiological or forensic studies.